Genetically expressed Förster resonance energy transfer (FRET) biosensors are a powerful tool to study the activation of signalling proteins and pathways in a live cell and in vivo setting. The prototypical RhoGTPases Rac1 and RhoA are temporally and spatially synchronized during cell protrusion; this tight control and dynamic regulation of activity is necessary for coordinated cell migration. We demonstrate an approach to simultaneous imaging of Rac1 and and RhoA biosensors in live tissue to study the spatiotemporal relationship between these two molecules. We use a custom multichannel fluorescence lifetime imaging (FLIM) system and employ a spectral-lifetime fitting approach to overcome the spectral overlap between the biosensors by exploiting the difference in lifetimes of the biosensor fluorophores. We fit our data to an analytical model of the lifetime and intensity of each biosensor to determine the abundance and activity of each biosensor across the field of view.
We have crossed a recently published ECFP-YFP Rac1 FRET biosensor reporter mouse with a EGFP-RFP RhoA FRET mouse to produce a dual reporter mouse. We demonstrate that we can readout the activation of Rac1 and RhoA in primary cells and tissues derived from the dual biosensor mouse. This approach allows us to directly visualise the mutually inhibitory response of RhoA and Rac1 on a subcellular level to treatment with small molecule inhibitors and activators.